title: Expression and gene knockdown of zebrafish Ca(2+)/calmodulin-dependent protein kinase Iδ-LL.
abstract: Ca(2+)/calmodulin-dependent protein kinase Iδ (CaMKIδ) is expressed ubiquitously, but little is known about its physiological functions. Recently, we cloned and characterized two splice variants of zebrafish (Danio rerio) CaMKIδ (CaMKIδ-S/L). In the present study we cloned a new CaMKIδ isoform, CaMKIδ-LL, encoded by a different gene from CaMKIδ-S/L. While the catalytic domain of CaMKIδ-LL showed 86% identity that of CaMKIδ-S/L, it had a unique C-terminal sequence. To clarify the functional role of CaMKIδ-LL, we investigated the biological significance of this new isoform during zebrafish embryogenesis. Although CaMKIδ-LL exhibited essentially the same catalytic properties and substrate specificities as the other CaMKIδ isoforms, it showed different temporal and spatial expression. During zebrafish embryogenesis, RT-PCR analysis detected CaMKIδ-LL expression after 48 h post-fertilization. Western blotting in adult zebrafish demonstrated that CaMKIδ-LL is expressed in the brain, the eye, and, abundantly, in fins. Knockdown of CaMKIδ-LL expression using morpholino-based antisense oligonucleotides resulted in an increase in abnormal embryos with small fins and underdeveloped cartilage. These phenotypes were rescued by co-injection with recombinant CaMKIδ-LL. These results clearly indicated that CaMKIδ-LL plays an important role in the generation of cartilage and fins during zebrafish embryogenesis.
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Senga
authors.forename: Keiko
authors.initials: K
authors.lastname: Yoshioka
authors.forename: Isamu
authors.initials: I
authors.lastname: Kameshita
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sueyoshi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1 / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D042541:Intracellular Space / Q000378:metabolism; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D015027:Zebrafish / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D020033:Protein Isoforms; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1
keywords: CaMKIδ; Embryogenesis; Gene knockdown; Protein phosphorylation; Zebrafish